The present invention relates generally to bacterial antigens and genes encoding the same. More particularly, the present invention pertains to the cloning, expression and characterization of the Mig Fc-receptor protein from several Streptococcus bacteria species, and the use of the same in vaccine compositions.
Mastitis is an infection of the mammary gland usually caused by bacteria or fungus. The inflammatory response following infection results in decreased milk yield as well as quality, and causes major annual economic losses to the dairy industry.
Among the bacterial species most commonly associated with mastitis are various species of the genus Streptococcus, including Streptococcus aureus, Streptococcus uberis (untypeable), Streptococcus agalactiae (Lancefield group B), Streptococcus dysgalactiae (Lancefield group C), Streptococcus zooepidemicus, and the Lancefield groups D, G, L and N streptococci. Some of those species are contagions (e.g. S. agalactiae), while others are considered environmental pathogens (e.g. S. dysgalactiae and S. uberis).
The environmental pathogen S. uberis is responsible for about 20% of all clinical cases of mastitis (Bramley, A. J. and Dodd, F. H. (1984) J. Dairy Res. 51:481-512; Bramley, A. J. (1987) Animal Health Nutrition 42:12-16; Watts, J. L. (1988) J. Dairy Sci. 71:1616-1624); it is the predominant organism isolated from mammary glands during the non-lactating period (Bramley, A. J. (1984) Br. Vet. J. 140:328-335; Bramley and Dodd (1984) J. Dairy Res. 51:481-512; Oliver, S. P. (1988) Am. J. Vet. Res. 49:1789-1793).
Mastitis resulting from infection with S. uberis is commonly subclinical, characterized by apparently normal milk with an increase in somatic cell counts due to the influx of leukocytes. The chemical composition of milk is changed due to suppression of secretion with the transfer of sodium chloride and bicarbonate from blood to milk, causing a shift of pH to a more alkaline level. S. uberis mastitis may also take the form of an acute clinical condition, with obvious signs of disease such as clots or discoloration of the milk and swelling or hardness of the mammary gland. Some cases of the clinical disease can be severe and pyrexia may be present. For a review of the clinical manifestations of S. uberis mastitis, see, Bramley (1991) Mastitis: physiology or pathology. p. 3-9. In C. Burvenich, G. Vandeputte-van Messom, and A. W. Hill (ed.), New insights into the pathogenesis of mastitis. Rijksuniversiteit Gent, Belgium; and Schalm et al. (1971) The mastitis complex-A brief summary. p. 1-3. In Bovine Mastitis. Lea and Febiger, Philadelphia.
Conventional antibacterial control methods such as teat dipping and antibiotic therapy are effective in the control of many types of contagious mastitis, but the environmental organisms typically found in all dairy barns are often resistant to such measures. Vaccination is therefore an attractive strategy to prevent infections of the mammary glands, and has been shown to be beneficial in the case of some contagious mastitis pathogens.
However, the literature is limited regarding vaccination studies with environmental pathogens such as S. dysgalactiae and S. uberis, and variable results have been observed. In some cases, immunization has resulted in increased sensitivity to the specific organism and in other cases strain-specific protection has been obtained.
For example, previous studies have shown that primary infection with S. uberis can considerably reduce the rate of infection following a second challenge with the same strain (Hill, A. W. (1988) Res. Vet. Sci. 44:386-387). Local vaccination with killed S. uberis protects the bovine mammary gland against intramammary challenge with the homologous strain (Finch et al. (1994) Infect. Immun. 62:3599-3603). Similarly, subcutaneous vaccination with live S. uberis has been shown to cause a dramatic modification of the pathogenesis of mastitis with the same strain (Hill et al. (1994) FEMS Immunol. Med. Microbiol. 8:109-118). Animals vaccinated in this way shed fewer bacteria in their milk and many quarters remain free of infection.
Nonetheless, vaccination with live or attenuated bacteria can pose risks to the recipient. Further, it is clear that conventional killed vaccines are in general largely ineffective against S. uberis and S. agalactiae, either due to lack of protective antigens on in vitro-grown cells or masking of these antigens by molecular mimicry.
The current lack of existing mastitis vaccines against S. agalactiae or the contagious streptococcus strains is due at least in part to a lack of knowledge regarding the virulence determinants and protective antigens produced by those organisms which are involved in invasion and protection of the mammary gland (Collins et al. (1988) J. Dairy Res. 55: 25-32; Leigh et al. (1990) Res. Vet. Sci. 49: 85-87; Marshall et al. (1986) J. Dairy Res. 53: 507-514).
S. dysgalactiae is known to bind several extracellular and plasma-derived proteins such as fibronectin, fibrinogen, collagen, alpha-II-macroglobulin, IgG, albumin and other compounds. The organism also produces hyaluronidase and fibrinolysin and is capable of adhering to and invading bovine mammary epithelial cells. However, the exact roles of the bacterial components responsible for these phenotypes in pathogenesis is not known.
Similarly, the pathogenesis of S. uberis infection is poorly understood. Furthermore, the influence of S. uberis virulence factors on host defense mechanisms and mammary gland physiology is not well defined. Known virulence factors associated with S. uberis include a hyaluronic acid capsule (Hill, A. W. (1988) Res. Vet. Sci. 45:400-404), hyaluronidase (Schaufuss et al. (1989) Zentralbl. Bakteriol. Ser. A 271:46-53), R-like protein (Groschup, M. H. and Timoney, J. F. (1993) Res. Vet. Sci. 54:124-126), and a cohemolysin, the CAMP factor, also known as UBERIS factor (Skalka, B. and Smola, J. (1981) Zentralbl. Bakteriol. Ser. A 249:190-194), R-like protein, plasminogen activator and CAMP factor. However, very little is known of their roles in pathogenicity.
The use of virulence determinants from Streptococcus as immunogenic agents has been proposed. For example, the CAMP factor of S. uberis has been shown to protect vertebrate subject from infection by that organism (Jiang, et al., U.S. Pat. No. 5,863,543).
The xcex3 antigen of the group B Streptococci strain A909 (ATCC No. 27591) is a component of the c protein marker complex, which additionally comprises an xcex1 and xcex2 subunit (Boyle, U.S. Pat. No. 5,721,339). Subsets of serotype Ia, II, and virtually all serotype Ib cells of group B streptococci, have been reported to express components of the c protein. Use of the xcex3 subunit as an immunogenic agent against infections by Lancefield Group B Streptococcus infection has been proposed. However, its use to prevent or treat bacterial infections in animals, including mastitis in cattle, has not been studied.
The group A streptococcal M protein is considered to be one of the major virulence factors of this organism by virtue of its ability to impede attack by human phagocytes (Lancefield, R. C. (1962) J. Immunol. 89:307-313). The bacteria persist in the infected tissue until antibodies are produced against the M molecule. Type-specific antibodies to the M protein are able to reverse the antiphagocytic effect of the molecule and allow efficient clearance of the invading organism.
M proteins are one of the key virulence factors of Streptococcus pyogenes, due to their involvement in mediating resistance to phagocytosis (Kehoe, M. A. (1991) Vaccine 9:797-806) and their ability to induce potentially harmful host immune responses via their superantigenicity and their capacity to induce host-cross-reactive antibody responses (Bisno, A. L. (1991) New Engl. J. Med. 325:783-793; Froude et al. (1989) Curr. Top. Microbiol. Immunol. 145:5-26; Stollerman, G. H. (1991) Clin. Immunol. Immunopathol. 61:131-142).
However, obstacles exist to using intact M proteins as vaccines. The protein""s opsonic epitopes are extremely type-specific, resulting in narrow, type-specific protection. Further, some M proteins appear to contain epitopes that cross react with tissues of the immunized subject, causing a harmful autoimmune response (See e.g. Dale, J. G. and Beachey, E. H. (1982) J. Exp. Med. 156:1165-1176; Dale, J. B. and Beachey, E. H. (1985) J. Exp. Med. 161:113-122; Baird, R. W., Bronze, M. S., Draus, W., Hill, H. R., Veasey, L. G. and Dale, J. B. (1991) J. Immun. 146:3132-3137; Bronze, M. S. and Dale, J. B. (1993) J. Immun 151:2820-2828; Cunningham, M. W. and Russell, S. M. (1983) Infect. Immun. 42:531-538).
Chimeric proteins containing three different fibronectin binding domains (FNBDs) derived from fibronectin binding proteins of S. dysgalactiae and Staphylococcus aureus have been expressed on the surface of Staph. Carnosus cells. In the case of one of these proteins, intranasal immunizations with live recombinant Staph. Carnosus cells expressing the chimeric protein on their surface resulted in an improved antibody response to a model immunogen present within the chimeric surface protein (Liljeqvist, S. et al. (1999) FEBS Letters 446:299-304).
Bacterial Fc receptors (surface moieties that bind to immunoglobulin molecules through a non-immune mechanism, i.e., to the Fc portion of the antibody) are a class of binding proteins further categorized by their reactivity with different classes and subclasses of mammalian immunoglobulins. The type I receptor (also known as Protein A), the most extensively studied and characterized, has been isolated from Staphylococcus aureus, and binds to IgG types 1,2 and 4; this receptor type further exhibits cross-reactivity with IgA and IgM. The type II Fc receptor, found on a few Lancefield Group A streptococci, and the type III receptor (also known as Protein G), found on the majority of human group C and group G strains of streptococcus, have been reported to react with all four types of IgG. In the case of the type III receptor, binding to IgG is highly specific; the protein does not cross-react with IgA or IgM. The type IV receptor is found in certain bovine group G streptococci, and the type V receptor is found on certain strains of Streptococcus zooepidemicus. The type VI Fc receptor has been isolated from S. zooepidemicus strains S212 and RSS-212, and binds rat IgG with high affinity, i.e., 100 times that of Protein A binding, and 30 to 40 times as great as Protein G binding. (Boyle, et al., U.S. Pat. No. 4,977,082). For a discussion of Fc receptors, see Langone (1982) Adv. Immunol. 32:167 and Myhre et al. (1984) Basic Concepts of Streptococci and Streptococcal Diseases (Holm and Christensen, eds.) Redbook Ltd., Chertsey, Surrey, England.
Utility for Fc binding proteins have to date been limited to antibody detection and purification. With respect to clinical applications, a method of extracorporeal blood treatment of autoimmune disease which employs Fc binding proteins to remove antigen-antibody complexes has been proposed (see e.g. Fahnestock, U.S. Pat. No. 4,954,618). However, their use in vaccine compositions has not previously been described nor suggested.
Until now, the protective capability of the S. dysgalactiae Mig protein against mastitis has not been studied, nor has the S. dysgalactiae Mig protein been isolated or characterized.
Accordingly, the present invention provides Fc receptor proteins and uses therefor. In one embodiment, the invention is directed to a vaccine composition comprising a pharmaceutically acceptable vehicle and an Fc receptor protein. In certain embodiments, the Fc receptor protein is selected from the group consisting of:
(a) a Streptococcus dysgalactiae Mig protein comprising the amino acid sequence shown at amino acid positions 1 to 669, inclusive, of FIGS. 1A-1D (SEQ ID NO:4);
(b) an Fc receptor protein having at least about 70% sequence identity to (a); and
(c) immunogenic fragments of (a) and (b), said fragments comprising at least about 5 amino acids.
In some embodiments, the vaccine composition comprises and adjuvant.
In yet further embodiments, the invention is directed to a method of producing a vaccine composition. The method comprises the steps of
(a) providing an Fc receptor protein or an immunogenic fragment thereof, the fragment comprising at least about 5 amino acids, and
(b) combining said protein with a pharmaceutically acceptable vehicle.
In another embodiment, the invention is directed to a method of treating or preventing a bacterial infection in a vertebrate subject. The method comprises administering to the subject a therapeutically effective amount of a vaccine composition as described above.
In certain embodiments the bacterial infection is a streptococcal infection. Additionally, the bacterial infection may cause mastitis.
In yet another embodiment, the invention is directed to a method of treating or preventing a bacterial infection in a vertebrate subject comprising administering to the subject a therapeutically effective amount of a polynucleotide that comprises a coding sequence for an Fc receptor protein.
In certain embodiments, the bacterial infection is a streptococcal infection and may cause mastitis.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.